Variation of alkaloid productivity among several clones of hairy roots and regenerated plants ofAtropa belladonna transformed withAgrobacterium rhizogenes 15834

Hairy root cultures ofAtropabelladonna were established by transformation withAgrobacterium rhizogenes 15834. Five clones of them were employed to study the production of hyoscyamine, the main constituent of the plant, together with other tropane alkaloids. The growth and alkaloid production of each clone were differently affected by basal liquid culture media tested. The transgenic plants regenerated from each clone of the hairy roots had different phenotypes and diverse alkaloid productivity both in the cultured condition and in productivitiy both in the cultured condition and in hydroponics.Abbreviations ANOVA analysis of variance - B5 medium Gamborg B5 medium - BA N⁶-benzyladenine - B.S. Balanced Solution - dw dry weight - EC electric conductivity - fw fresh weight - GC/MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MS medium Murashige and Skoog medium - NAA naphthalene-l-acetic acid - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - TMS trimethylsilyl - WP medium Woody Plant medium     

Promotion of flowering and morphological alterations in Atropa belladonna transformed with a CaMV 35S-rolC chimeric gene of the Ri plasmid

Summary Kanamycin-resistant plants of belladonna (Atropa belladonna) were obtained after Agrobacterium mediated transformation. When a rolC gene, which is one of the loci located on Ri plasmid of Agrobacterium rhizogenes, was co-introduced with a kanamycin resistant (NPT II) gene under control of a cauliflower mosaic virus 35S promoter, the rolC gene was expressed strongly in leaves, flowers, stems and roots. The transformed plants exhibited dramatic promotion of flowering, reduced apical dominance, pale and lanceolated leaves and smaller flowers. On the other hand, when native rolC gene was co-introduced with NPT II, the transgenic plants obtained did not exhibit the altered phenotypes observed in 35S-rolC transformants, and the expression level of the rolC gene was much lower than in 35S-rolC transformants. These results suggest that the morphological changes in transgenic Atropa belladonna were related to the degree of expression of the rolC gene.Abbreviations native rolC rolC gene under control of its own promoter - 35S-rolC rolC gene under control of a cauliflower mosaic viras 35S promoter     

Tropane alkaloid production by Agrobacterium rhizogenes transformed hairy root cultures of Atropa baetica Willk. (Solanaceae)

Atropa baetica hairy root cultures were induced after infecting stem segments with Agrobacterium rhizogenes strain ATCC 15834. Accumulation of the tropane alkaloids atropine and scopolamine by hairy roots cultured in half- and full-strength Murashige and Skoog (MS) medium was high, although this was not growth associated. These alkaloids were also released into both liquid media. Higher tropane alkaloids present both in hairy roots and liquid medium occurred in half MS medium, showing a clear relationship between slow growth of cultures and higher product accumulation. The pH of both nutrient media varied as culture progressed, and seemed to be associated with the release of scopolamine. GC-MS analyses showed the presence of a new compound, namely tigloylpseudotropine; moreover, 3α-isobutyryloxytropane, formerly found only in plant leaf tissue, was also identified in the hairy roots. Received: 18 August 1997 / Revision received: 30 November 1997 / Accepted: 20 January 1998     

Intertribal hybrid cell lines of Atropa belladonna (X) Nicotiana chinensis obtained by cloning individual protoplast fusion products

Summary After fusion of isolated mesophyll protoplasts of belladonna (Atropa belladonna) with callus protoplasts of Chinese tobacco (Nicotiana chinensis) followed by mechanical isolation and cloning of individual heteroplasmic fusion products, 13 cell clones were obtained. The hybrid nature of most of the clones has been confirmed by biochemical (studies of amylase isozymes), cytogenetic (size and morphology of chromosomes) and physiological (peculiarities of cell-growth in vitro) analyses. Study of chromosomes and isozyme patterns in the hybrid cell lines revealed the presence of both parental genomes, without an indication of chromosome elimination, six months after hybridization. In 4 cell lines shootlike structures and plantlets have been produced by means of transfer to organogenesis-inducing media. The data obtained are interpreted as new evidence for the possibility of using non-sexual hybridization for the production of intergeneric, intertribal plant hybrids which cannot be obtained by sexual crossing. From these results the potential of Atropa (X) Nicotiana hybrids as a model system for genetic studies of distantly related plant species is discussed.This work is part of a joint project between Institute of Botany of the Ukrainian Academy of Sciences, Kiev, USSR, and Institute of Pharmaceutical Biology, University of Munich, FRG     

Regeneration of flax plants transformed by Agrobacterium rhizogenes

Regeneration of flax (Linum usitatissimum) following transformation by either Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector, or Agrobacterium rhizogenes carrying an unmodified Ri plasmid, was examined. Hypocotyl and cotyledon explants inoculated with A. tumefaciens formed transformed callus, but did not regenerate transformed shoots either directly or via callus. However, cotyledon explants inoculated with A. rhizogenes formed transformed roots which did regenerate transformed shoots. Ri T-DNA encoded opines were detected in the transformed plantlets and Southern hybridization analysis confirmed the presence of T-DNA from the Ri plasmid in their DNA. Transformed plantlets had curled leaves, short internodes and some had a more developed root system characterized by plagiotropic behaviour.     

Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA

Single and multiple infections of carrot discs were carried out with Agrobacterium strains harbouring different segments of pRi1855 TL-DNA cloned in the binary vector Bin 19 and with a strain carrying the TR-DNA from the same Ri plasmid. Roots induced by the various co-inoculations were cultured and their growth patterns were followed. Abundant roots could be induced by TL-DNA rol genes A, B and C as a single insert (rolA+B+C) and by rolB alone provided an extended segment beyond its 5 noncoding region was included in the construction. A depression of rooting capability was caused by the inclusion of rolC together with rolB (rolB+C). In all cases co-inoculation with the Agrobacterium carrying TR-DNA-borne auxin genes was necessary for root induction since none of the rol constructions was in itself capable of eliciting any response; an exceeding majority of these roots were however shown to contain rol genes but no TR-DNA. Rooting was also elicited if rol constructions were co-inoculated with a strain carrying TL-DNA genes 13 and 14 (ORF13+14) instead of the TR-DNA strain. These roots were shown to contain both rol genes and ORF13+14. Striking differences in growth properties were shown by roots containing different complements of TL-DNA genes. Typical hairy root traits, high growth rate, branching and, most noticeably, absence of geotropism, were shown by roots containing rolB alone, while roots with rolA+B+C were geotropic as normal carrot roots. Hairy root traits were conferred to rolA+B+C roots by the concomitant presence of ORF13+14 and by the addition of auxin to the culture medium. A model is presented which attempts to rationalize the growth patterns by assigning interplaying roles to the various TL-DNA genes involved.     

Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna

The activities of enzymes related to the biosynthesis of N-methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine -Nmethyltransferase and -N-methylornithine decafboxylase were undetectable, indicating that -N-methylornithine is an unlikely intermediate in the formation of N-methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from dl-[5-¹⁴C]ornithine, l-[U-¹⁴C]arginine, [U-¹⁴C]agmaine and [1,4-¹⁴C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, dl--difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, dl--difluoromethylornithine. Together with the demonstration that label was incorporated from [U-¹⁴C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from dl-[ 5-¹⁴C] ornithine and l-[U-¹⁴C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.Abbreviations DFMA dl--difluoromethylarginine - DFMO dl--difluoromethylornithine We thank Miss E. Bent for valuable technical assistance and J. Eagles, K. Parsley and Dr. F. Mellon for mass-spectrometric analysis. We are grateful to Dr. A.J. Parr and Dr. M.J.C. Rhodes for helpful discussions. We are indebted to the Merrell Dow Research Institute, Cincinnati, Ohio, USA for supplying DFMA and DFMO.     

Production of tropane alkaloids by transformed root cultures of Atropa belladonna in stirred bioreactors with a stainless steel net

A scale up of transformed root cultures of Atropa belladonna from a 300-ml flask to a 30-l tank was accomplished without any reduction in alkaloid productivity. Cutting treatment of seed cultures showed no distinct effect on root growth, morphology, and alkaloid content in conical flasks during 1 month of culture. Randomly cut roots thus grown were further cultivated in 3-l and 30-l modified stirred bioreactors for a scale-up culture. After 1 month of culture, 1490 mg of tropane alkaloids was produced by a 30-l culture of A. belladonna transformed roots. These roots contained the same level of atropine (5.4 mg/ g dw) as the roots of this plant grown in the field for 12 months and still contained a considerable amount of other alkaloids including 1.6 mg/g dw of 6-β-hydroxyhyoscyamine, 0.9 mg/g dw of scopolamine, and 2.0 mg/g dw of littorine. Received: 12 June 1998 / Revision received: 31 August 1998 / Accepted: 27 October 1998     

Direct regeneration of transformed plants from stem fragments of potato inoculated with Agrobacterium rhizogenes

Mannopine and cucumopine strains of Agrobacterium rhizogenes were used for genetic transformation in two cultivars of potato (Solanum tuberisum L.). An overnight pretreatment of internodes with -naphthaleneacetic acid prior to bacterial infection was found to strongly inhibit shoot formation. On the contrary, infection with bacterial strains enhanced the frequency of shoot formation, compared with the controls, except for the strain 15834 which completely inhibited shoot formation in both potato cultivars. Shoots developed directly from the upper part of both inoculated and control explants, at a frequency ranging from 1 to 5 shoots per fragment. Among 93 shoots regenerated, 9 were found to be opine positive, and exhibited an altered phenotype with shortened internodes. Histological study revealed that the transformed shoots developed directly from cells of the internode sections, and not from induced roots. When grown in an insect-proof tunnel, the transformed plants had both altered and normal phenotypes and were able to produce tubers.Abbreviations df degree of freedom - F F distribution - MS Murashige and Skoog basal medium - NAA -naphthalenacetic acid - p probability - T-DNA transferred DNA     

Characterization of ornamental Datura plants transformed by Agrobacterium rhizogenes

Summary Datura arborea and D. sanguinea hairy roots were produced by cocultivation of leaf fragments with Agrobacterium rhizogenes strain NCPP 1855. Adventitious buds emerged spontaneously, without exogenous growth regulators, from seven hairy root clones of D. arborea and from one hairy root clone of D. sanguinea. Regenerated plants were successfully acclimatized in the greenhouse. The integration of the bacterial TL-DNA into the genome of the putative transformed plants was confirmed by Southern blot analysis. Transgenic plants displayed increased ability to root in vivo. Morphological traits with relevant ornamental value like plant height, leaf number, size and shape, internode number, and internode length were also affected. Transformation by wild-type Ri TL-DNA provided the chance to study plant growth and differentiation and to select improved genotypes.     

The use of Agrobacterium rhizogenes transformed roots to obtain transgenic shoots of the apple rootstock Jork 9

The apple rootstock Jork 9 was transformed using four different Agrobacterium rhizogenes virulent strains. The mannopine strain 8196 gave the best results in the production of chimeric plants compared to two agropine strains (A4 and 15834) and one cucumopine strain. Shoot regeneration was performed on both untransformed and transformed roots. Optimum combination and concentration of thidiazuron (TDZ) and -naphtaleneacetic acid (NAA) was different between untransformed and transformed roots. From the transformed roots seven shoots were obtained and propagated as individual clones. All shoots from these clones rooted on a hormone-free medium contrary to untransformed shoots that did not root under similar culture conditions. Differences in the morphology of the leaves and stems were observed between the clones. The transformed status of the different clones was verified with mannopine tests, PCR and Southern blot analyses. Five clones contained the mas1', the ORF 13 and the rolB genes, whereas two clones contained only the rolB gene.     

TL-DNA from Agrobacterium rhizogenes plasmid pRi1855 reduces the osmotic pressure in transformed plants grown in vitro

Growth, water content, osmotic pressure and solute content were examined for normal potato (Solanum tuberosum L. cv. Desiree) and a derivative (line D9X8a), which was genetically transformed with T_L-DNA from Agrobacterium rhizogenes. Plants were grown (i) in vitro, (ii) in a growth chamber and (iii) in the field. In vitro, the transformed potato plants produced more biomass than the untransformed plants, partly because they had a higher water content. Potassium concentration and osmotic pressure were lower in cell sap extracted from the transformed potato shoots. In some cases the difference was as much as 50%. These differences were less clear, absent or reversed in plants from a growth chamber or from the field. In the field, however, transformed potato senesced early. It is suggested that a cellular basis for these observations may be changes induced by Ri T_L-DNA expression products in plant membrane properties.Abbreviations Ri root inducing - Ti tumour inducing - T-DNA transferred DNA     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype

Summary Segments of the TL-DNA of the agropine type Ri plasmid pRi 1855 encompassing single and groups of open-reading frames were cloned in the Ti plasmid-derived binary vector system Bin 19. Leaf disc infections on Nicotiana tabacum led to transformed plants, some of which showed typical hairy root phenotypes, such as the wrinkled leaf morphology, excessive and partially non geotropic root systems and the ability of leaf explants to differentiate roots in a hormone-free culture medium. Particularly interestingly, most of these traits were shown by plants transformed with a TL-DNA segment encompassing the single ORF 11, corresponding to the rolB locus. Hairy root can be induced by this latter T-DNA segment on wounded stems of tobacco plants; hairy root induction on carrot discs requires, on the contrary, a more complex complement of TL-DNA genes.Abbreviations YMB yeast mannitol broth - MS Murashige and Skoog medium - 6-BAP 6-benzylaminopurine - NAA naphthalene acetic acid - Km kanamycin - Cb carbenicillin     

AgNO_3对颠茄毛状根生物碱及关键酶基因表达的影响

为了研究不同浓度AgNO_3对颠茄毛状根生长过程中托品烷类生物碱及影响其合成关键酶基因表达的影响,该研究使用五种不同浓度的AgNO_3处理黑暗培养12 d的颠茄毛状根,2 d后收集毛状根并测定其鲜、干质量、托品烷类生物碱的含量、部分生理指标(MDA、Pro、可溶性糖、可溶性蛋白)、关键酶基因(pmt、trI、h6h)表达量。结果表明:AgNO_3虽然抑制了颠茄毛状根的生长,但却促进了托品烷类生物碱的积累,与对照相比,50、100、150μmol·L~(-1)处理均显著性地提高了托品烷类生物碱的产量。同时在150μmol·L~(-1)处理下,毛状根代谢途径中MDA与对照相比均具有显著性的提高,脯氨酸含量在150μmol·L~(-1)下也有了显著性提高,达到了5.92μg·g~(-1)FM,是对照的2.91倍。可溶性糖与可溶性蛋白含量在150μmol·L~(-1)浓度下处理时其含量分别是对照的1.55倍和1.67倍。托品烷类生物碱合成过程中关键酶基因(pmt、trI、h6h)的表达量在不同浓度AgNO_3处理下均有不同程度的提高。由此可以推断,AgNO_3在150μmol·L~(-1)处理下,可能通过调控脯氨酸、可溶性蛋白、可溶性糖这几种初级代谢产物或某几种关键酶基因的表达来影响颠茄毛状根托品烷类的合成。     

Regeneration and characterization of plants from potato root lines transformed by Agrobacterium rhizogenes

Summary Transformed root lines were obtained after infection of leaf segments and tuber discs of tetraploid potato cvs Bintje and Desirée with Agrobacterium rhizogenes. In response to shoot induction, about 10% of the root lines produced shoots through callus formation. The tests for opine suggest that all 26 shoot lines of cv Bintje (Ri-Bintje) and 13 of Desirée (Ri-Desirée) were transformed. All shoot lines were tetraploid except for one octoploid subshoot line of cv Desirée; no aneuploids were observed. With the exception of two shoot lines derived from the same root line, all other Ri-Bintje plants showed a pattern of phenotypic variation, generally observed among transformed plants. In contrast, the phenotype of Ri-Desirée plants was uniform and normal; variation was observed in tuber form and size. Phenotypic variation observed among Ri-plants appeared to be mainly root line-dependent, particularly for height of plants and tuber size and form. Variation was also observed within root and shoot lines and was more pronounced among the Ri-Bintje plants. Segregation of phenotypic characteristics was observed among transformed plants, resulting in the occurrence of phenotypes resembling the control. Chromosomal stability and the frequent reversion to normal phenotype of Ri-plants make A. rhizogenes particularly suitable as a virulence vector in the binary transformation system for the transfer of desirable genes.     

Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.     

The non-conserved region of cucumopine-type Agrobacterium rhizogenes T-DNA is responsible for hairy root induction

The T-DNA regions of three strains of Ri plasmids 1855, 8196, 2659 (agropine, mannopine and cucumopine type respectively) share two highly conserved regions flanking a non-homologous central part [1,2]. We have cloned segments of the cucumopine Ri plasmid 2659 T-DNA in the binary vector system Bin 19 and infected carrot discs with recombinant Agrobacterium strains. We show here that the central non-conserved region is crucial in hairy root induction as it is sufficient to induce rooting on the apical (auxin-rich) surface of carrot discs; in order to observe rooting on the basal (auxin-depleted) side of the discs, a longer T-DNA fragment, also encompassing part of the right conserved region, had to be utilized in conjunction with a Agrobacterium strain carrying aux genes. Differences of growth properties in culture are exhibited by roots transformed with different fragments of pRi 2659 T-DNA, although all transformed roots show the plagiotropic behaviour typical of hairy roots.     

Use of Agrobacterium rhizogenes to create transgenic apple trees having an altered organogenic response to hormones

Summary The apple rootstock, M26, was genetically and phenotypically transformed using the Agrobacterium wild-type strain, A4. First, chimeric plants were obtained having transformed roots and normal aerial parts. Transformed plants were then produced through regeneration from transformed roots. Transformation was demonstrated by molecular hybridization and opine analysis. The effects of hormones on organogenesis was altered in transformants: cytokinins were required to form roots, whereas auxin was toxic at the concentration used to induce rooting in the control.